​​Growing interest by corn growers in the USA to having seed tested for germination at temperatures typical of actual planting dates has resulted in several questions.  Many of these issues have been raised by seed producers for many years.  Why does some seed within a seed lot germinate and others not?  What causes some seed to not germinate under cold conditions although the warm test result shows they are alive?  What should be the minimum acceptable cold test result?
 
Hybrid corn is mostly bred to produce a large amount of grain per area of soil.  Most of that volume and weight is carbohydrate deposited in the endosperm. Genetics affecting the production and storage of carbohydrate is mostly biologically separate from the utilization of that stored carbohydrate into metabolism needed for growth of the plant.  A hybrid in which maximum grain production per plant occurs because the combination of genetics from a specific male and female parent are a match.  Those two inbreds, however, frequently differ in vulnerabilities to environments that affect germination.  The inbred that becomes the female plant in hybrid seed production becomes the genetic source of some cellular components such as the mitochondria and chloroplasts.  Mitochondria are especially significant in deriving the energy from carbohydrates to drive the metabolism needed for production of new cells during the germination process. This is probably the main biological reason that seed companies have identified certain parents as more reliable for germination.
 
Membrane systems, including those within mitochondria, are essential to cellular function.  Cellular membranes are also vulnerable to damage, especially during the rehydration after seed has be dried.  The damage is independent of temperature.  Movement of water through the pericarp into the embryo cells at the same rate in warm wet soil as in cold soil.  Sudden swelling of the membrane-bound organelles causes some breakage.  Remaining metabolism in undamaged portion repairs the damaged membranes.  This rate of repair is temperature dependent.  Repair is slower at lower temperatures.  If soil temperature is below 55°F, it is believed that very little imbibition damage to cellular membranes especially those of the mitochondria will occur.  In the field with low temperatures, the damaged seed and slow growth becomes vulnerable to invasion by microbes that can further inhibit seedling emergence.
 
Cold germination testing is intended to identify the percent of seed within a sample that have sufficient membrane damage to inhibit or delay germination and consequently not become a productive plant.  Developing reliable, repeatable test methods is a challenge resulting in multiple lab differences in results.  Some variability is due to subtle aspects of temperature of water, nature of germination media and definition of damaged seedlings usually called ‘abnormals’.  Added to the biological-environmental variables are the difficulties in adequately sampling. The initial source causing the imbibitional chilling damage vulnerability could have been disease or moisture stress in the production field, handling in the harvest, drying and bagging process.  Genetics, especially of the female parent also interact with all of these factors.  In many ways it is amazing that we produce mostly high germinating seed. (Corn Journal 4/24/2018)

​While we wait for the first signs of emergence from the soil with the coleoptile poking up to the light, the real action has been happening within the cells of the shoot and roots of the seed embryo. It is cell division and cell elongation that pushes the shoot tissue up and root down. That action is occurring as the cell organelles are activated

​It is difficult to imagine 32000 genes distributed among the 10 chromosomes in the nucleus of a single cell within the embryo of the corn seed. But the microscopic cell also contains many other substances that contribute to cell function once it is activated with germination. Proteins and lipids contribute to the function of the outer plasma membrane surrounding the cell, but membrane-like structures also are intertwined within the cells. Endoplasmic reticulum is used to transport cell products. Ribosomes are attached to the outside of ‘rough’ endoplasmic reticulum. These ribosomes are the organelles in which RNA codes, originating from the DNA, are used to link the amino acids to form proteins. Adjacent endoplasmic reticulum is used to transport the newly formed proteins to sites in the cell appropriate for that protein’s function.

Mitochondria, independent organelles within the cell, are the site of transferring glucose molecules in the chemical energy used by other cell functions. These organelles, carried along in the egg cell from the maternal parent plant, have their own DNA for genetics but are dependent on the rest of the cell and nuclear DNA to provide the glucose, proteins and lipids for structure and function. This symbiotic relationship is in all animal, plant and fungal species, originating a few billion years ago and certainly is significant in corn performance. Mutations in the mitochondria DNA are the source of cytoplasmic male sterility, at least partly because of a genetic defect in the outer membrane of the mitochondria results in defective pollen production. Integrity of the membrane of the endoplasmic reticulum, ribosomes, mitochondria, nucleus and outer cell membrane after imbibition of water is essential to that early activation of the seed embryo and the emergence that we anticipate.

​ 
Each seed in a single cross hybrid corn bag is slightly different in its biological and even genetic history.  Rarely are the two inbred parents in a seed field 100% homozygous plus some of those plants may have their own mutations.  Fortunately, drastic mutants are usually caught by the seed producer before pollination. An individual female plant in the seed field may produce 200-300 seed. And one 80000 kernel bag of seed corn must represent seed from 300-400 individual plants.
 
We want to think of all single cross seed in a bag are the same, but they are not identical genetically or in germination quality.  Even with multiple generations of selfing in development of the parent seed, some mutations occur with each generation of seed increase prior to planting in the hybrid seed field.  Most often these mutations are non-consequential to hybrid performance and especially not visible in the field where many small environmental effects are affecting appearance of the plants.  The closer we get to discerning DNA differences the more difficult it becomes to distinguish inconsequential differences from the drastic ones.
 
Seed quality differences among the seed in that one bag of hybrid seed also shows differences.  A warm test may show 95% germination but even beyond the non-germinating ones, there will be some that are slower to germinate than others even when all environment is uniform.   As the percent germinated gets lower, more late ones become evident.  A cold test, especially like ours at Professional Seed Research, Inc. in which we cover the seed with 3/4 inch of artificial soil, nearly always show lower percent germination and more late emerging plants than the warm test.  Why are all the seed not with the same quality when produced in same field?
 
Unfortunately, not all the seed on a single seed field ear have the same environment. First silk, coming from the ovules near the base of the ear, emerge 3-6 days before the final silk.  Successful pollination by the correct male parent is dependent of many variables, including factors associated with maturity for the male and female in the seed field.  In general, pollen timing is affected more by accumulation of heat units whereas silking is favored by water.  Cool wet pre-flowering weather can lead to silks being exposed before pollen.  This not only makes the seed more vulnerable to contamination by outside pollen from field corn, resulting in outcrosses, but also to infection from fungi such as Fusarium or Diplodia species traveling down the silk channel before it closes after pollination.  The opposite can happen with hot dry weather, in which the silk emergence is delayed, causing the pollen to be spent before all the silk emerges.  Consequently, there often are differences in germination (and outcrosses) among seed positions on the ear.  

We humans have adapted a tropical plant (Teosinte) and created a species adapted to temperate zone called Zea mays. Along the way, selections were made to allow germination of seed and early growth and at very non-tropical, low temperatures. It also requires careful seed production, storage and planting.

Hybrid corn is mostly bred to produce a large amount of grain per area of soil. Most of that volume and weight is carbohydrate deposited in the endosperm. Genetics affecting the production and storage of carbohydrate is mostly biologically separate from the utilization of that stored carbohydrate into metabolism needed for growth of the plant. A hybrid in which maximum grain production per plant occurs because the combination of genetics from a specific male and female parent are a match. Those two inbreds, however, frequently differ in vulnerabilities to environments that affect germination. The inbred that becomes the female plant in hybrid seed production becomes the genetic source of some cellular components such as the mitochondria and chloroplasts. Mitochondria are especially significant in deriving the energy from carbohydrates to drive the metabolism needed for production of new cells during the germination process. This is probably the main biological reason that seed companies have identified certain parents as more reliable for germination.

Membrane systems, including those within mitochondria, are essential to cellular function. Cellular membranes are also vulnerable to damage, especially during the rehydration after seed has be dried. The damage is independent of temperature. Movement of water through the pericarp into the embryo cells at the same rate in warm wet soil as in cold soil. Sudden swelling of the membrane-bound organelles causes some breakage. Remaining metabolism in undamaged portion repairs the damaged membranes. This rate of repair is temperature dependent. Repair is slower at lower temperatures. If soil temperature is below 55°F, it is believed that very little imbibition damage to cellular membranes especially those of the mitochondria will occur. In the field with low temperatures, the damaged seed and slow growth becomes vulnerable to invasion by microbes that can further inhibit seedling emergence.

Cold germination testing is intended to identify the percent of seed within a sample that have sufficient membrane damage to inhibit or delay germination and consequently not become a productive plant. Developing reliable, repeatable test methods is a challenge resulting in multiple lab differences in results. Some variability is due to subtle aspects of temperature of water, nature of germination media and definition of damaged seedlings usually called ‘abnormals’. Added to the biological-environmental variables are the difficulties in adequately sampling. The initial source causing the imbibitional chilling damage vulnerability could have been disease or moisture stress in the production field, handling in the harvest, drying and bagging process. Genetics, especially of the female parent also interact with all of these factors. In many ways it is amazing that we produce mostly high germinating seed.

​Each plant of a single-cross hybrid has the identical genetics, including those affecting the speed of seedling growth.  Uniform seed quality and environment will allow uniform environment. Hybrids differ in the speed of emergence but uniformity of emergence within a field becomes an important component of maximum grain yield.
 
Corn embryo’s being planted on May 1 are about ¼ inch (0.6 cm) in length. The future shoot portion of the embryo is half the size of the embryo. Two to three months later that shoot length has been multiplied by 800-1000.  Within the embryo are cells with organelles such as mitochondria, plastids, ribosomes and other membranous structures needed to carry out this remarkable growth rate. Within the nuclei of these cells are the 10 pairs of chromosomes with the 30-40000 genes, coded by long strings of nucleic acids.   Within a few hours of water imbibition, the few genes in the mitochondria are activated. Appropriate codes within their DNA produce RNA strings of nucleic acid, that are moved to ribosomes, producing proteins appropriate to enzymatically remove the energy binding the carbon and oxygen molecules in glucose and moving that energy into adenosine triphosphate (ATP).  This energy is utilized in manufacturing the other structures for rapid cell elongation and cell duplication, pushing the seedling shoot to the soil surface.
 
With exposure to light, some cellular plastids with guidance from their own DNA and supplies from the other cell components, produce chlorophyll. This pigment allows absorption of light frequencies providing energy to drive the capture of carbon, oxygen and hydrogen molecules in the process of photosynthesis.  Resulting glucose molecules are moved to the growing cells that utilize the new molecules in manufacture of structural complex molecules such as fatty acids and proteins used for cell metabolism and cell wall structures such as cellulose and lignin.

 
It is easy to be amazed when we seed the rapid growth of young corn plants and even more impressed to know that we are only seeing the result of remarkable interactions occurring at the cellular level.  
 

​I find it interesting to realize that much of what we see in a corn plant from germination to maturity is happening out of the sight of most of us. After germination, the root shoot heads downwards and the shoot grows upwards, regardless of orientation of the seed in the soil. Plant hormones auxins, cytokinins and gibberellins are primary in affecting growth directions of these tissues. Those interactions are affecting the cell growth in those tissues, especially whether they elongate or not. The affects do not really end with germination.

Cytokinins and auxins are operative during all of the corn plants life, including the movement of sugars to the young kernels. These two kinds of hormones have different roles in origin and effect on corn growth. Cytokinins are mostly produced in root tips in root meristems and transported through the water distribution in the xylem tissue. Auxins are mostly produced in stem meristems and distributed in the phloem system. Cytokinins are associated with increasing cell division in the stem meristems whereas auxins are involved in cell elongation. Apical dominance resulting in the corn plant usually having only one upright stem is because of the interactions of the auxins produced in the apical meristem. Removing that stem tip in early corn development and thus reducing auxin production tips the balance towards more cytokinin and stimulation of cell division in the lateral buds of the corn plant, resulting in branches.

Pollination of the multiple ovules in the corn ear results in attraction of cytokinins to each developing kernel. Moisture stress during the first 10 days after pollination is known to cause early death to some kernels, perhaps because of reduction transportation of cytokinins to the most immature embryos (my conjecture!). Cell division in the new embryo meristems establishes the movement of sugars through the phloem to the kernels. Much of the sugar is deposited into the endosperm portion where it is changed to more complex carbohydrates and thus allow the osmotic pressure for more sugar movement towards the kernels.

More is known about the effect of these plant hormones on plant growth than all of the mechanisms involved with those effects. Auxins involvement in cell growth involves softening cell walls, making elongation of cells easier. Cytokinins have been shown to prevent protein breakdown and activating protein synthesis.

Cytokinins produced in root meristems are transported to and stimulate the cell division in the kernel embryos. Meristems of those embryos produce auxins. Auxins are associated with production of ethylene which has been associated with formation of abscission tissue as leaves and fruit mature. It is assumed that the auxins are associated with formation of the black layer at the base of kernels, resulting in stoppage of movement of material to the kernels.

We know that these plant hormones are associated with the growth of corn tissues including the formation of kernels but there remains lots to learn of the actual molecular interactions that allows this to happen. Meanwhile, corn breeders, agronomists and growers attempt to coordinate it all by selecting the genetics that maximize grain production. AND, new knowledge is coming as those interested in the science continue to research into the processes that most of us witness but don’t see the minutia at the cellular level.

​Corn seed is planted in complex environment with a mix of soil particle density, moisture levels and other organisms, some of which capable of digesting any carbohydrates they can reach. When seed does not emerge as expected we often look for a cause including fungi associated with the poorly emerging seed. This often is not simple.

Among the perplexing interactions in corn is that with corn and the fungus Fusarium verticilloides. This fungus was formerly known as Fusarium moniliforme and is the asexual stage of the fungus Gibberella fujikuroi. Many, (most?) seed samples germinated by paper methods will show at least a few seeds with this fungus growing from them, even with the appearance of normal seed germination. The fungus does produce a toxin called fumonisin that can cause rejection of grain by some livestock and grain elevators. This can occur in kernels showing not symptoms of infection.

The fungus can be found in corn roots, stalks and leaves as well, often without symptoms. It is not unusual to find this species growing from dead corn leaf tissue when moistened. It is as if it is an inhabitant of corn. Does it become transmitted to the next generation from infected seed? It is acknowledged that seed can be infected via growth of the fungus in the silk. Or does it become from infected debris in the soil? Or, perhaps entering thru injuries to plant tissue.

A study published at Appl Environ Microbiol. 2003 Mar; 69(3): 1695–1701 followed the spread of F. verticilloides from infected seed and from inoculated soil through corn plants using a carefully designed group of experiments using fluorescent strain of the fungus detected by fluorescent microscopy. They confirmed that this fungus can infect through the root to the mesophyll, often growing between cells. This can result in stunted seedlings, especially if grown under low light conditions. This growth can advance into the stalk tissue, in leaves and even into the seeds, sometimes without showing symptoms. More of the fungus was found in the plant if the soil inoculum load was increased. There was spread to the roots and to the rest of the plant from infected seed but the soil inoculum load appeared to be more significant.

Low light association with increased symptoms suggests that the metabolic health of the plant affects the defense against this fungal invasion. That also is consistent with the presence of this fungus in nearly all rotted stalks. If other stalk rotting fungi, such as those associated with Diplodia stalk rot, Gibberella stalk rot or Anthracnose stalk rot are not found, we tend to call it Fusarium stalk rot because it is always there. The low physiological state of stressed corn plants as they approach completion of grain fill increase the vulnerability the expansion of this fungus into the dead stalk tissue.

Microbes inhabiting the corn plant reflect the complexity that actually is affecting the biology of the corn plant.

​As corn seed is moved from a dry environment of storage encouraging slow metabolism to the wet, more complex environment of moist soil, germination and emergence becomes our next measure of success. 
 
It is difficult to sort out the real cause of seed not emerging or emerging much later than adjacent plants.  Seeds are planted in environments that vary every few inches for water holding capacity, organic content and microbes.  Furthermore, each individual seed varies slightly in its cellular membrane status.  With imbibition causing swelling of the membrane bound cell contents, some seed can have problems getting effective metabolism for early cell growth to push out the root and stem structures.
 
Cell metabolism includes producing the response to attacks by potential pathogens in the soil. These anti-pathogen chemicals (phytoalexins) are usually produced with a complex system of detecting the microbe and concentrating the phytoalexin into the area of the attack.  Weakened seed not only are likely to release more carbohydrates and proteins into soil because of membrane injury, but also be less capable of responding to the microbes invading root and mesocotyl tissue.
 
Diagnosis of seedling disease becomes complicated also.  Pathologists can isolate a fungus such as a Fusarium species or an oomycete like a Pythium species, but the actual cause probably involves some interaction between the microbes, metabolic quality for the ‘diseased’ seedling, and a complex environment not only providing potential pathogens but also affecting the seedlings metabolic rate. Soil organisms are affected by the environments as well.  Leakage of carbohydrates directs their growth toward the seedling roots but temperatures favor some over others.  Pythium’s swimming spores do well in cool wet environments but can be inhibited by certain seed treatments that have very little effect on fungi such as Fusarium species.  Other seed treatments can inhibit the latter group of microbes but are less effective against Pythium.  Corn seed genetics and seed quality can be greater factors than either group of chemicals.  Cold wet heavy soils for a prolonged time can overcome all methods of defense.
 
After the stress on the seedlings is reduced, remaining plants that emerge can give normal production especially if they are uniform in growth with adjacent corn plants.  The metabolism of these plants will promote the recovery and normal root growth.  Those plants that survive but emerge later than adjacent plants will have difficulty competing for light and mineral uptake which will be reflected in grain productivity.
 
Life for the corn plant, like for the rest of us, becomes more complex after we start living in our environments.